Image-Guided Hydrogen Gas Delivery for Protection from Myocardial

Article

Image-guided hydrogen gas delivery for protection from myocardial ischemia-reperfusion injury via microbubbles Yingjuan He, Bo Zhang, Yihan Chen, Qiaofeng Jin, Junru Wu, Fei Yan, and Hairong Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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ACS Applied Materials & Interfaces

Image-guided hydrogen gas delivery for protection from myocardial ischemia-reperfusion injury via microbubbles

Yingjuan He †, 1, Bo Zhang ‡, 1, Yihan Chen§, Qiaofeng Jin †, Junru Wuφ, Fei Yan †,

§,*

and

Hairong Zheng †,§

†

Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and

Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055 China ‡

Department of Echocardiography, Shanghai Eastern Hospital Affiliated to Tongji University,

Shanghai 200120 China §

Department of Ultrasonography, The Third Affiliated Hospital of Southern Medical

University, Guangzhou 510500 China φ

1

*

Department of Physics, University of Vermont, Burlington, VT 5405-0160

These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: [email protected].

Address: Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Shenzhen 518055 China Tel: +86 755 86392284

Fax: +86 755 96382299

1

ACS Paragon Plus Environment


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Abstract Cardiomyocyte death induced by ischemia-reperfusion is a major cause of morbidity and mortality worldwide. Hydrogen (H2), as an antioxidant, has been shown to have great potentials in preventive and therapeutic applications to the lethal injury that occurs on ischemia-reperfusion. However, H2 is sparingly soluble in water, resulting in its poor bioavailability in blood and damaged tissues. Here, we have developed an ultrasound-visible H2 delivery system by loading H2 inside of microbubbles (H2-MBs) to prevent myocardial ischemia-reperfusion injury. Using this system, the concentrations of H2 in unit volume can be greatly improved under normal temperature and pressure conditions. H2-MBs can be visually tracked with ultrasound imaging systems and can effectively release the therapeutic gas. In vivo systemic delivery of H2-MBs in myocardial ischemic rats at the start of reperfusion resulted in a significant reduction of infarct size and pathological remodeling. Further analysis showed that this approach markedly inhibited cardiomyocyte apoptosis and reduced myocardial inflammation and oxidant damage in myocardial ischemia-reperfusion rats. These results indicate that H2-MBs are a promising visual delivery system for H2-based therapeutic applications. Keywords: Hydrogen Gas, Microbubbles, Myocardial Ischemia-Reperfusion Injury, Ultrasound imaging, Drug delivery

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1. INTRODUCTION Myocardial ischemia-reperfusion injury has been widely accepted as a stimulus for tissue destruction and possible cardiac failure.1 When one or more branches of the coronary arteries are narrowed or occluded, the heart is deprived of oxygenated blood, and the subsequent restoration of perfusion and reoxygenation frequently results in exacerbated tissue injury.2 Pathologically, a potential mechanism for ischemia-reperfusion injury is the overproduction of reactive oxygen species (ROS) in the damaged myocardial tissue, especially during the first few minutes of reperfusion. ROS production triggers the opening of the mitochondrial permeability-transition pore and results in a wide range of pathological processes, including various cell death programs, microvascular dysfunction and the inflammatory cascade.3-5 To date, numerous attempts using therapeutic gases such as nitric oxide, carbon monoxide and hydrogen sulfate have been used to attenuate myocardial ischemia-reperfusion injury.6-9 Most of these techniques have shown promise in the laboratory but have shown less encouraging results in clinical applications. A key issue arises from the inherent toxicity of these gases.10 Recently, hydrogen gas (H2), one of the most well-known molecules, was proposed to have potential in preventive and therapeutic applications against organ dysfunction induced by ischemia-reperfusion injuries.11-15 The effectiveness of H2 against oxidative stress-induced disorders in other models has also been widely demonstrated.16-21 Compared with other therapeutic antioxidants, H2 has a number of unique advantages, including (i) the selective reduction of hydroxyl radicals, the most cytotoxic of ROS, and the effective protection of cells without disturbing metabolic oxidation-reduction reactions or disrupting other ROS involved in cell signaling and other physiological roles;11 (ii) 3



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penetration of biomembranes and diffusion into the cytosol, mitochondria and nucleus (its rapid gaseous diffusion might make it highly effective in reducing cytotoxic radicals); (iii) more importantly, H2 is a safe gas without obvious side effect on human.22, 23 Conventionally, the following three strategies are used to deliver H2 in therapeutic applications: (i) straightforward inhalation of H2,12, 16, 24 (ii) drinking hydrogen dissolved in water

18, 25, 26

or (iii) intravenous/intraperitoneal injection of hydrogen-saturated saline.27

However, the effects from all delivery strategies are dependent on the solubility of H2 in water, saline or blood. Unfortunately, H2 is sparingly soluble in aqueous solvents, resulting in its poor bioavailability in blood and damaged tissues. In addition, the flammability of H2 brings great hardships and safety hazards when fabricating H2-containing formulations. Furthermore, no image-guided H2 delivery system is currently available for use in biomedical applications. These limitations have provided an incentive to search for new, safe H2-delivery systems for effective therapeutic use. Microbubbles (MBs) are small, gas-filled microspheres. For a long time, MBs have been highlighted due to their clinical application as contrast-enhancing agents in ultrasound diagnosis. Beyond this role, MBs are important drug delivery vehicles that can stably carry drugs in the blood circulation and that have many therapeutic applications.28 Drugs can be loaded onto the MB shell, embedded in the shell matrix, or loaded into the internal void.29 Recently, documents using MBs as vehicles to encapsulate therapeutic gas have been reported.30 Here, we introduce and demonstrate the potential of H2-loaded MBs (H2-MBs) as an antioxidant for the prevention of myocardial ischemia-reperfusion injury in a rat model.

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2. MATERIALS AND METHODS 2.1. Preparation of H2-MBs. Fifteen milligrams of phospholipids composed of 1,2-distearoyl-sn-glycero-phosphocholine (DSPC, purchased from Avanti Polar Lipid. Inc) and 1,2-distearoyl-sn-glycero-3-phosphonethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt, DSPE-PEG2000, purchased from Avanti Polar Lipid. Inc) at a molar ratio of 90:10 were mixed in chloroform. The solvent was evaporated with a steady nitrogen stream to form a thin film on a glass tube. The phospholipid films were then placed under vacuum for approximately 2 h to completely remove the solvent. The dried phospholipid films were then hydrated at 60°C with 5 ml of buffer solution containing glycerol, 1,2-propylene glycol and 0.1 M Tris buffer at a volume ratio of 10:10:80, followed by sonication until the lipid films were completely dispersed. Subsequently, the sonicated phospholipid suspensions were transferred to 4-ml glass vials that were sealed with a cap (approximately 1 ml each vial), and the gases in the vials were displaced with octafluoropropane (C3F8) using a self-designed device. The vials containing the phospholipid suspensions and C3F8 were mechanically vibrated using oscillators (MONITEX capsule Mixer, revolution 4,500 rpm) for 45 sec, and the control microbubbles (vehicle) were obtained.31 To fabricate the H2-MBs, a given amount of H2 was injected into the inverted vials via a 1-ml syringe, and the heavier C3F8 gases were squeezed out through another needle. These vials were mechanically vibrated, as stated above, to obtain the H2-MBs. 2.2. Measurement of the H2 release profiles. The H2 release profiles were measured in PBS and blood using a needle-type H2 sensor (Unisense) according to the previous study.12 Briefly, 0.2 ml of the H2-MBs or control MBs (vehicle) was added to 5 ml of PBS. The H2 release 5



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profiles were then determined by immersing a needle-type H2 sensor into the MB suspensions and recording the data in a real-time manner. For the in vivo H2 release profiles, the needle-type H2 sensor was inserted in the LV cavity (arterial blood) of five healthy rats, and 2 ×1010 H2-MBs or vehicle were administered into the rats through intravenous injection. The concentration of H2 in the blood was then continuously recorded. 2.3. Ultrasound imaging. Ultrasound imaging of the H2-MBs was performed using a small-animal-dedicated,

high-resolution

ultrasound

imaging

system

(Vevo

2100;

VisualSonics, Toronto, Canada) or using an IE33 ultrasound machine (Philips Medical Systems, MA, USA). For the in vitro linear B-mode ultrasound imaging experiments, various concentrations (5×105/ml, 5×106/ml, 5×107/ml, 5×108/ml or 5×109/ml in PBS) of the H2-MBs were added into translucent bags; PBS was used as a control. The bags were then immersed in a water bath, and ultrasound imaging was performed using a Vevo 2100 Imaging System with the following parameters: probe, MS-400; gain, 25 dB; focal depth, 4～6 mm; transmit power, 30%; mechanical index, 0.2; dynamic range, 60 dB, frame rate, 25 Hz; and center frequency, 40 MHz. For the in vivo contrast enhanced ultrasound imaging experiments, three healthy rats were anesthetized by inhalation with 1.5% isoflurane, anchored in a supine position, and then used for the in vivo ultrasound imaging experiments. H2-MBs (2 ×1010 bubbles per animal) were intravenously administered into the rats, and the H2-MBs in LV cavity of hearts were imaged using the Vevo 2100 Imaging System with the following parameters: probe, MS-250; gain, 20 dB; focal depth, 21～23 mm; transmit power, 10%; mechanical index, 0.2; dynamic range, 40 dB, frame rate, 17 Hz; and center frequency, 18 MHz. For detecting H2-MBs which were delivered into the myocardial tissue, the hearts were imaged using a Philips IE33 ultrasound machine with the following parameters: probe, S5-1; gain, 44 dB; focal depth, 1～3 cm; dynamic range, 50 dB, frame rate, 40Hz and a center 6


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frequency of 3 MHz. The signal intensities were quantified by QLAB software.

2.4. Myocardial ischemia–reperfusion model. All of the animal experiments were carried out in compliance with the relevant laws and institutional guidelines for the care and use of laboratory animals. The protocols were approved by the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Science. Briefly, eight-week-old female Sprague-Dawley rats were anesthetized with 10% chloral hydrate (0.3 ml/kg body weight) by intraperitoneal injection; additional doses were given as needed to maintain anesthesia. Regional myocardial ischemia was then induced by ligating the left anterior descending coronary artery with a 5-0 silk suture. Ischemia was confirmed by ECG S-T segment elevation, and after 30 min of ischemia, the vehicle or H2-MBs were intravenously administered via tail vein injection. The reperfusion was reestablished for 3 or 24 h and the coronary artery was retied. Some rats were randomly chosen to be analyzed for the area at risk by injection of Evans blue dye after producing myocardial infarction.

2.5. Assessment of the infarct size. To assess the infarct size, the hearts were harvested, sliced into 5 sections and then incubated with 0.1% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Sigma-Aldrich) at 37°C for 20 min. The slices were photographed and weighed. Non-infarcted viable myocardium stained red and the infracted tissue remained unstained (white), followed by measuring with computed planimetry (ImagePro Plus version 6.1). Infarct size was calculated as [(A1 × W1) + (A2 × W2) + (A3 × W3) + (A4 × W4) + (A5 × W5)], where A is the area of infarct for the slice and W is the weight of the respective section. Infarct size was expressed as a percentage of the total heart weight for control hearts and H2-MB-treated hearts as described previously.32 2.6. Echocardiography. After 30 min of ischemia and 24 h of reperfusion, 2 days later, the rats were anesthetized by inhalation with 1.5% isoflurane. The animals were anchored to a 7



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positionable platform in a supine position, and short axis echocardiography was accomplished with a Vevo 2100 system using a MS-250 probe (sweep speed 1000 Hz, Frequency 21 MHz). The LV dimensions and FS% and EF% were obtained using M-Mode in the parasternal long axis view, from the trailing edge to the leading edge. Echocardiographic variables were calculated according to the American Society of Echocardiography (ASE) guidelines.33

2.7. H&E staining and immunohistochemistry. The rats were anesthetized with sevoflurane after 30 min of ischemia and 24 h of reperfusion, after which the hearts were dissected and fixed with 4% buffered formalin at 4°C for 24 h. The hearts were then paraffin-embedded and cut into 5-µm thick sections, and the sections were deparaffinized, rehydrated in PBS using standard procedures, and stained with hematoxylin and eosin (H&E staining kit, Sigma-Aldrich). Subsequently, the sections were incubated with rabbit anti-rat caspase-3 monoclonal antibody (1:400 dilution; Cell Signaling) at 4°C overnight, and following three washes in PBS, the sections were incubated with goat anti-rabbit IgG secondary antibody (1:1000 dilution; Abcam) for 30 min at room temperature. Immunoreactive signals were visualized using DAB. The sections were then counterstained with hematoxylin and examined under a microscope (Q500MC, Leica image analysis system).

2.8. TUNEL assays. TUNEL assays were performed using an in situ cell death detection kit according to the product instructions. In brief, the heart sections were analyzed using digoxigenin-labeled dUTP that was catalytically incorporated into the DNA by terminal deoxynucleotidyl transferase. The stained sections were photographed and the number of TUNEL-positive nuclei was counted in a blinded manner.

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2.9. ELISA detection. Heart tissues were homogenized as described in the Supplementary Methods. The TNF-α, IL-1βand 8-OHdG levels were then measured in the supernatants which were prepared as stated above using commercially available kits (eBioscience, Minneapolis, MN, USA) according to the manufacturer's instructions. The absorbance of the samples was read using a microplate reader (Multifunctional Enzyme Mark Synergy4, BioTek, USA) at a wavelength of 450 nm, and the concentrations of TNF-α, IL-1βand 8-OHdG were calculated according to the standard curves generated using the OD values of standard solutions of TNF-α, IL-1β and 8-OHdG. The quantitative measurement of caspase-3 activity was performed using a caspase-3 colorimetric assay kit (Sigma, St. Louis, MO, USA), from which the absorbance at 405 nm was determined and the concentration was calculated. 2.10. Measurement of malondialdehyde (MDA) and MPO. The MDA and MPO levels in myocardial tissue were determined using MDA and MPO kits (Nanjing Jianchen Biological Institute, China). Briefly, tissue homogenates were prepared using the above-described methods, and the MDA and MPO protein concentrations were determined using the BCA Protein Assay with BSA as a standard. The MDA (nmol/mg) and MPO levels (U/mg) were normalized to the total proteins.

2.11. Quantification of ROS in myocardial tissue. After 30 min of ischemia and 40 min of reperfusion, the animals were euthanized, the hearts were removed, and heart samples were homogenized. The homogenates were then immediately reacted with 10 µM of 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein

diacetate

acetyl

(CM-H2DCFDA,

Probes),

5

purchased

from

Molecular

µM

ester of

diaminofluorescein-2-diacetate (DAF-2DA, purchased from Daiichi Pure Chemicals Co.) or 5 µM of nitroblue tetrazolium (NBT) for 30 min to detect cellular H2O2, NO· or O2-·levels, 9



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respectively. We then measured the fluorescent signals for CM-H2DCFDA and DAF-2DA using an automatic microplate reader (BioTekTM Synergy4) at 510 nm, with excitation at 490 nm. The absorbance due to the reduction of NBT to NBT diformazan by O2-·was detected at 550 nm. We used 0.4 µM of 3'-(p-hydroxyphenyl) fluorescein (HPF, purchased from Molecular Probes) to detect ·OH, and the fluorescent signal was measured at 515 nm, with excitation at 490 nm.

2.12. Statistical analysis. The data are expressed as the means ± SEMs and were analyzed using SPSS 13.0 (Chicago, IL, USA). An unpaired two-tailed Student’s t-test for single comparisons and an analysis of variance (ANOVA) for multiple comparisons were performed. A value of P < 0.05 was considered statistically significant.

3. RESULTS 3.1. Characterization of H2-MBs. This procedure was firstly developed to load H2 into MBs through the following steps, including film formation/hydratization, vial sealing, octafluoropropane (C3F8) displacement, H2 displacement and vibration (Figure 1a). The characteristic differences of MBs with different C3F8/H2 ratios (vol/vol) were compared and found that the concentrations of the MBs decreased with increasing addition of H2. Small amount of MBs were produced in the absence of C3F8 (C3F8/H2 = 0:3) (Figure S1), with an approximately 100-fold lower MB yield compared with other groups (Supplementary, Table 1). Typically, the H2-MBs showed a polydisperse and spherical morphology under a microscope (Figure S2). No significant differences in the mean MB diameters, size distributions and microbubble stability with the use of C3F8/H2 ratios of 3:0 and 1:1, whereas 10


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the MB size increased in the absence of C3F8 (Figure S3, S4 and Table S1). The H2 content in the H2-MBs was 1.46 ± 0.08 and 0.14±0.13 µmol H2 per ml MB suspension when the C3F8/H2 ratios of 1:1 and 0:3 were used (Table S1). Therefore, the formulation (the ratio of C3F8/H2 = 1:1) was used in all subsequent in vitro and in vivo studies. Next, the H2 release profiles was analyzed in vitro and in vivo by monitoring the time-course of the changes in H2 levels using a needle-shaped hydrogen sensor electrode that was inserted directly into the H2-MB suspension in PBS or into the rat left ventricular cavity. Results showed that the H2 level in PBS started inceased after H2-MBs were added and reached a maximum level after 2 min (180 ± 0.02 µmol/l) (Figure 1b). The incremental rate of H2 release in animals was similar to that observed in PBS, with a maximum of 119 ± 0.02 µmol/l 2 min after intravenous injection of H2-MBs. By contrast, the rate of decay of the H2 concentration was faster in vivo, with an H2 level of 9.02 ± 0.02 µmol/l after 10 min (Figure 1c). The H2 levels did not change in vitro or in vivo after the addition of the control MBs (vehicle containing pure C3F8). 3.2. H2-MBs can be visualized by ultrasound imaging. Because MBs are the most commonly used contrast agents for contrast-enhanced ultrasound imaging, the imaging effects of H2-MBs in vitro and in rat hearts were examined. Results demonstrated that H2-MBs caused an effective echogenic response, and the intensity of the ultrasound signal was concentration-dependent (Figure 2a). Quantitative analysis revealed the intensities of the ultrasound signals were 43.01 ± 1.29, 57.86 ± 1.45, 88.29 ± 0.76, 114.68 ± 0.74 and 161.04 ± 1.48 dB for 1×105, 1×106, 1×107, 1×108 and 1×109 bubbles per milliliter of H2-MBs, respectively (Figure 2b). In addition, the imaging effects from the pre-injection or 11



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post-injection of H2-MBs into the left ventricular (LV) of rat hearts was also compared, showing a significant enhancement of the ultrasound signals after H2-MB injection as comparison with the pre-injection group (Figure 2c). Quantitatively, the signal intensity rapidly increased after the H2-MBs were injected, and the signal reached a maximum level of 123.39 ± 6.34 dB and then decreased to 87.86 ± 8.12 dB after 5 min (Figure 2d). Also, we found H2-MBs can deliver into the rat myocardial tissue (Figure 2e). The ultrasound signal intensity of the myocardial tissue after injection of H2-MBs had a 2.83-fold increase when compared with that of the myocardial tissue before injection of H2-MBs (P < 0.01) (Figure 2f). A high-power destruction pulse was able to destroy the H2-MBs and resulted in a dramatic decrease of the ultrasound signal intensity, confirming the presence of H2-MBs in the myocardial tissue (Figure S5). 3.3. H2-MBs limit myocardial infarct size. We then investigated whether H2-MB doses have different effects on infarct size after myocardial ischemia-reperfusion injury. Rats were subjected to 30 min of ischemia and 24 h of reperfusion. Ischemia was confirmed by electrocardiogram (ECG) S-T segment elevation after left coronary artery (LCA) occlusion (Figure S6), and the H2-MBs or vehicles were administered into the tail veins at low-dose (4×109 bubbles) or high-dose (2×1010 bubbles) concentrations before reperfusion. Representative cross-sections of vehicle- or H2-MB (high-dose)-treated rats are shown in Figure 3a. Evaluation of infarct size revealed a dose-response effect, with high-dose H2-MBs displaying more significant cytoprotection than low-dose H2-MBs (P < 0.01) (Figure 3b). Rats receiving high-dose H2-MBs displayed an infarct size of 12.81 ± 3.21% compared with vehicle-treated rats (INF/LV: 42.67 ± 2.60%), representing a 69.97% reduction in infarct size. 12


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By contrast, rats receiving low-dose H2-MBs showed less significant cytoprotection, with an infarct size of 26.21 ± 3.10%, which only represented a 38.57% reduction in infarct size. We also examined the protective effects of high-dose H2-MBs on rats that underwent 30 min of ischemia and 3 h of reperfusion, and the evaluation of infarct size revealed a 28.06% reduction in infarct size (P < 0.05) (Figure 3c). 3.4. H2-MBs preserve LV structure and function. To determine the impact of H2-MBs on pathological LV remodeling, LV morphology and function were monitored using echocardiography after 30 min of LCA ischemia and 24 h of reperfusion. In comparison to sham-treated rats, vehicle-treated rats showed maladaptive pathological remodeling after myocardial infarction. Notably, administration of H2-MBs before reperfusion reduced the deterioration of pathological remodeling after myocardial infarction (Figure 4a). Moreover, rats treated with H2-MBs showed no significant increase in the left ventricular end-diastolic dimension (LVEDD) or left ventricular end-systolic dimension (LVESD) compared with the sham-treated group. By contrast, rats treated with vehicle displayed significantly increased LVESD and LVEDD values after myocardial ischemia–reperfusion (Figure 4b). Furthermore, vehicle-treated rats were found to have 21.68% and 15.81% decreases in ejection fraction and in the fractional shortening after myocardial ischemia compared with sham-treated rats, whereas rats receiving H2-MBs displayed minimal 8.44% and 7.21% reductions in ejection fraction and fractional shortening, respectively (Figure 4c). 3.5. H2-MBs reduce cardiomyocyte apoptosis. The myocardial structures of sham-, vehicleand H2-MB-treated rats were then histologically examined. Blinded histological analyses of heart sections stained with H&E were scored 24 h after reperfusion, and we found that rats 13



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receiving H2-MBs displayed a reduced degree of myocardial necrosis and hemorrhaging compared with rats receiving vehicle (Figure 5a, upper row). To elucidate whether H2-MBs reduce cardiomyocyte apoptosis, TUNEL assays and immunohistochemical staining were used to analyze rat heart tissues. We found that TUNEL-positive nuclei (green) were rarely found in the sham-treated animals, in contrast to the many more TUNEL-positive nuclei (35.53 ± 5.89%) that were observed in heart sections of rats after myocardial ischemia–reperfusion with vehicle MBs. Groups receiving H2-MBs before reperfusion displayed a significant reduction in the number of TUNEL-positive nuclei (8.62 ± 3.71%) compared with the vehicle-treated group, indicating less cardiomyocyte apoptosis in these heart sections (P < 0.01) (Figure 5a, middle row and 5b). Immunohistochemical staining with anti-caspase-3 antibody further confirmed lower caspase-3 expression in the H2-MBs-treated rat hearts than in vehicle-treated rat hearts. Quantitatively, an approximately 43.63% reduction in caspase-3 activity in H2-MB-treated rats was observed compared to the vehicle-treated group (Figure 5a, lower row and 5c). 3.6. H2-MBs reduce myocardial inflammation and oxidant damage. ELISA analysis was used to examine the key pro-inflammatory and oxidant-stress cytokines that were produced after 30 min of ischemia and 24 h of reperfusion. The results revealed that vehicle-treated rats had a significant increase in all examined cytokines compared with sham animals (P < 0.05). In contrast, H2-MB-treated rats had various degrees of reduced myocardial inflammation and oxidant damage. A 28.0% decrease in MPO for rats receiving H2-MBs was observed in comparison with vehicle-treated animals (P < 0.05) (Figure 6a), and the assessment of TNF-α and IL-1β levels in myocardial lysate revealed 39.0% and 14.5% reductions in H2-MB-treated 14


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rats compared to the vehicle-treated group, respectively (P < 0.01) (Figure 6b and 6c). In addition, ELISA analysis for pro-oxidant cytokines in myocardial homogenates revealed that rats receiving H2-MBs had significantly reduced 8-OHdG levels compared with vehicle-treated animals (115.78 ± 10.88 vs 88.31 ± 6.34 ng/mg proteins) (P < 0.01) (Figure 6d). The levels of myocardium malonaldehyde (MDA) in the H2-MB-treated group displayed a similar change, with levels 12.16% lower than those in the vehicle-treated group (P < 0.05) (Figure 6e). 3.7. H2-MBs selectively reduce hydroxyl radicals. Other studies have demonstrated that H2 can selectively reduce cytotoxic oxygen radicals and neutralize hydroxyl radicals (·OH) in living cells [11]. Therefore, we further investigated whether H2-MBs could effectively reduce ROS in the AAR of myocardium tissue after 30 min of ischemia and 40 min of reperfusion. H2-MB-treated rats displayed a significant reduction in the ·OH level after 40 min of reperfusion when compared to vehicle-treated rats (P < 0.05) (Figure 6f); however, the assessment of NO·, O2-·and H2O2 levels in myocardial lysates revealed no differences between any of the groups (P > 0.05) (Figure 6 g-i). 3.8. H2-MBs have good biocompatibility. Toxicological analyses were performed to detect whether H2-MBs are toxic to healthy rats. The analysis of blood, including complete blood counts, indicated that the blood count values were within the normal range 3 or 7 days after H2-MB injection by intravenous administration (Table S2). Histology was also performed on the heart, spleen, liver, lung, and kidney, and the results showed that H2-MBs did not exert adverse effects on these organs (Figure S7). Furthermore, no abnormal behaviors were observed in the rats injected with H2-MBs, demonstrating that H2-MBs have good 15



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biocompatibility and are nontoxic to rats.

4. DISCUSSION Currently, no image-guided H2 delivery system that can be used to protect from cardiovascular diseases is currently available. In this study, we designed H2-MBs for use in an ultrasound-guided therapeutic H2 delivery system to facilitate real-time and visual monitoring of therapeutic cargo delivery to myocardial tissue. This delivery system has the potential to establish a foundation for translating therapeutic gas-based therapies from basic science to clinical applications. In our current study, we found that there was a higher H2 content per unit volume of solution when using MBs to encapsulate H2 (approximate 3-fold higher at C3F8/H2 = 1:1) than when using H2-saturated saline (to dissolve H2 in saline under 0.4 MPa pressure),17 implying that H2-MBs can greatly improve the solubility of H2 per unit volume of solution. This moiety has the potential to easily achieve a higher H2 concentration in a limited-volume solution by increasing the number of H2-MBs rather than by increasing the pressure or volume. Furthermore, in our in vitro and in vivo studies, we found that H2-MBs can effectively release H2, resulting in a maximum H2 level of 180 ± 0.02 µmol/l in PBS and 119 ± 0.02 µmol/l in rat arterial blood, respectively. Notably, this was a nearly 6- to 10-fold higher level than what was observed in a previous study where rats were forced to inhale H2 gas.11 In their study, evidences have demonstrated hydrogen have a direct effect on oxidants by selectively reducing cytotoxic oxygen radicals and 20 ng H2 per ml arterial blood (equal to 10 µmol/l concentration) was considered to be effective therapeutic dose. Data from ultrasound imaging suggested that H2-MBs could be used for imaging under acoustic excitation. This unique acoustic property of MBs facilitates real-time tracking of H2 delivery to myocardial tissue and monitors the dose of the therapeutic gas because the 16


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ultrasound signal intensities are proportional to the concentrations of H2-MBs. To date, there have been no reports of image-guided H2 delivery systems for therapeutic applications using H2. Furthermore, the in vitro and in vivo imaging analyses in our study consistently suggested that the concentrations of H2-MBs were proportional to the ultrasound signal intensities (Fig. 2). Data from the assessment of myocardial infarct size and echocardiography indicated that H2-MBs could effectively protect against myocardial ischemia-reperfusion injury and thereby preserve LV function. We then assessed the effects of H2-MBs on myocardial apoptosis and inflammation after infarction. Histological analysis revealed a significant decrease in hemorrhaging and apoptosis, as well as a decrease in the expression of caspase-3 within the ischemic zone of rats received H2-MBs. In addition, the quantitative analysis of inflammatory cytokines revealed the myocardial levels of MPO, TNF-α and IL-1βto be markedly reduced when compared to those observed in vehicle-treated rats. In fact, the induction of cardioprotection through the anti-inflammatory effect of H2 has been previously reported.34 Additionally, our data revealed that the administration of H2-MBs reduced the levels of 8-OHdG and MDA in the ‘at risk’ area for infarction, which indicates that the antioxidant action of H2-MBs, at least partially, is directed by the ROS scavenging effect. Further analysis of ROS confirmed the H2-MBs effect of neutralizing toxic ROS. However, the levels of ·OH, but not of NO·, O2-· or hydrogen peroxide (H2O2), decreased in myocardial tissue from rats receiving H2-MBs after myocardial ischemia-reperfusion injury. These results are in accordance with previous work.11 In biological systems, the hydroxyl radical acts as the most reactive product of the ROS that are generated from superoxide anion through the Haber–Weiss reaction or from H2O2 through the Fenton reaction.35, 36 Thus, developing smart H2-MBs as a platform has a promising future for being used in medical applications as safe and effective antioxidants with minimal side effects. 17



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5. CONCLUSION In summary, we developed a novel, image-guided, therapeutic H2 delivery system that is simple to use, and the resulting H2-MBs greatly improved the solubility of H2 per unit volume of solution. More importantly, the H2-MBs could be visually tracked and used to protect from myocardial ischemia–reperfusion injury. Our study provides a potential solution to the bottleneck in the clinical translation of H2-based therapeutic applications.

ASSOCIATED CONTENT Supporting Information Six figures showing the produced MBs, microscopic image, size distributions, ultrasound image and signal intensity curve of rat heart, representative electrocardiogram of rats before and after LCA ischemia, histology of heart, liver, spleen, lung, kidney. Two tables showing size, concentration and H2 content of H2-MBs, and complete blood count of experimental rats before and after injection of H2-MBs.

AUTHOR INFORMATION Corresponding Author *(F.Y.) Tel: +86 755 86392284. E-mail: [email protected]. Author Contributions Y.H. and B.Z. contributed equally. F.Y. conceived and designed the experiments. Y.H., B.Z., Y.C., D.Z., and H.Y. performed the experiments. F.Y. and H.C. analyzed the data. F.Y., Y.H., J.W., and H.Z. cowrote the paper. All authors discussed the results and commented on the manuscript. Notes 18


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by the National Key Basic Research Program of China (973 Program) (Grant Nos. 2013CB733804, 2014CB744502), and the National Natural Science Foundation of China (Grant Nos. 81571701, 81371563, 11534013, 11325420, 81527901, 81571963).

Natural

Science

Foundation

of

Guangdong

Province

(Grant

No.

2014A030312006), Shenzhen Science and Technology Innovation Committee (Grant No. JCYJ20150521144321010).

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Figure legends: Figure 1. Preparation and characterization of H2-MBs. (a) Schematic illustration of the preparation of the H2-MBs. Fifteen milligrams of phospholipid mixture composed of DSPC and DSPE-PEG2000 (molar ratio = 90:10) was dissolved in chloroform. After film formation and hydration, the aqueous dispersions (liposomes) were bottled and sealed in 4-ml vials (1 ml each), and the air in the vials was displaced with octafluoropropane (C3F8). Subsequently, the vials were inverted, and a given amount of H2 was injected with a syringe. Thus, equal volumes of C3F8 were squeezed out of the vials. The H2-MBs were obtained after vibration for 45 s. (b) Representative image of the H2-MBs obtained from the above-mentioned method (at a ratio C3F8/H2 ratio of 1:1). (c-d) Representative time courses of the H2 release in vitro and in vivo. In the in vitro experiments, 4 ×109 H2-MBs or control MBs (vehicle) were added to 5 ml of PBS, and a needle-type H2 sensor was used to detect H2 release from the MBs. In the in vivo experiments, 2×1010 H2-MBs or vehicle were intravenously injected into rats, and the concentration of H2 in the blood was recorded continuously using a needle-type H2 sensor which was inserted into the left ventricular (LV) cavity (n = 5). Arrows indicate the time when the H2-MBs or vehicle was added.

Figure 2. Ultrasound signal detection of the H2-MBs. (a) Ultrasound images of H2-MBs dispersed in PBS at various concentrations from 5×105/ml, 5×106/ml, 5×107/ml, 5× 108/ml to 5×109/ml. Imaging was performed using a Vevo 2100 ultrasound imaging system. (b) Quantitative analysis of the ultrasound signal intensity of the H2-MBs in PBS. (c) Ultrasound images of the H2-MBs in left ventricular of rat hearts before and after tail-vein

25



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injection of H2-MBs. Imaging was performed using a Vevo 2100 ultrasound imaging system (n = 3). Upper row, pre-injection of H2-MBs; Bottom row, post-injection of H2-MBs; Left panel, B-mode images; right panel, contrast-mode images; LV, left ventricular (dotted cycle). (d) Time-intensity curves, for 300 seconds after the H2-MBs were injected into the rats. Arrow indicates the time of MBs injection. (e) Ultrasound images of the H2-MBs delivery into the rat myocardial tissue. Imaging was performed using a Philips IE33 ultrasound machine. Left, pre-injection of H2-MBs; right, post-injection of H2-MBs; LV, left ventricular (red cycle); IVS, interventricular septum (blue cycle); RV, right ventricular (green cycle). Arrows indicate the H2-MBs delivery into the rat myocardial tissue after tail-vein injection of H2-MBs. (f) Quantitative analysis of the ultrasound signal intensity of the H2-MBs (n = 3). **P < 0.01. The data represent the means ± s.d.

Figure 3. H2-MBs prevent myocardial infarction. (a) Representative TTC-stained, serial myocardial sections from rats treated with vehicle (left) or H2-MBs (right, high-dose). Tissue stained positive (red) represents viable myocardium. Myocardium that is not stained positive (white) represents the infarcted tissue. (b) Quantitative assay of the myocardial infarct sizes of sham-treated, vehicle-treated or H2-MB-treated rats (low-dose, 4×109 bubbles per rat; high-dose, 2×1010 bubbles per rat) after 30 min of LCA ischemia and 24 h of reperfusion. H2-MB-treated rats displayed a significant reduction in INF/LV (n = 7). *P < 0.05, **P < 0.01. INF: infarct area. The data represent the means ± s.d. (c) Quantitative assay of the myocardial infarct sizes of sham-treated, control-MB-treated or H2-MB-treated rats after 30 min of LCA ischemia and 3 h of reperfusion; 2 ×1010 H2-MBs were used for each H2-MB-treated rat. A 26


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similar protective effect was found (n = 5). **P < 0.01. The data represent the means ± s.d.

Figure 4. H2-MBs reduce adverse LV remodeling. (a) Representative M-mode echocardiographic images of rats receiving sham-operated (sham), ischemia–reperfusion with vehicle

or

ischemia–reperfusion

with

H2-MBs.

Measurements

of

the

M-mode

echocardiographic images of each group were performed 48 h after ischemia–reperfusion. Imaging was performed by using a Vevo 2100 system equipped with MS-250 scanhead probe at a 1000 Hz sweep speed and 21 MHz frequency. Scale bar (vertical), 4 mm. (b) Based on the echocardiographic images, the LV dimensions, including the left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD), were determined (n = 5). **P < 0.01. The data represent the means ± s.d. (c) The fractional shortening (% FS) and ejection fraction (% EF) were significantly improved in H2-MB-treated rats compared with the control group (n = 5). **P < 0.01. The data represent the means ± s.d.

Figure 5. H2-MBs protect the myocardial structure and reduce cardiomyocyte apoptosis. (a) Histological analysis of heart sections from sham-, vehicle- or H2-MB-treated rats that underwent 30-min of ischemia and 24 h of reperfusion; the H&E staining of these sections is shown (upper row). Rats receiving vehicle displayed obvious hemorrhaging and necrosis within the ischemic zone (upper row, middle), but myocardial damage was attenuated in the sections from rats treated with H2-MBs (upper row, right). TUNEL staining was performed to detect the apoptosis of cardiomyocytes (middle row), and a significant increase in the number of TUNEL-positive cells could be observed in the rats receiving vehicle (middle row, middle)

27



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compared with sham-treated rats (middle row, left). Rats receiving H2-MBs displayed a significant decrease in the number of TUNEL-positive nuclei (middle row, right). Immunohistochemical staining for caspase-3 expression was performed (bottom row), which showed relatively higher caspase-3 expression in the rats receiving vehicle (bottom row, middle) than that of sham-treated rats (bottom row, left). Decreased caspase-3 expression could be observed in rats receiving H2-MBs (bottom row, right); bar = 50 µm. (b) Plots of the apoptotic index was performed by calculating the percent of TUNEL-positive nuclei in five high-magnification fields, which demonstrated a significant reduction in the number of TUNEL-positive nuclei in H2-MB-treated rats compared with the vehicle group (n = 5). **P < 0.01. The data are the means ± s.d. (c) Caspase-3 expression was quantitatively analyzed using a caspase-3 colorimetric assay kit (n = 5). **P < 0.01. The data are the means ± s.d.

Figure 6. ELISA analysis of myocardial pro-inflammatory and pro-oxidant cytokines and reactive oxygen species. Heart tissue homogenates were obtained from sham-, vehicleor H2-MB-treated rats that had undergone 30 min of ischemia and 24 h of reperfusion to analyze the produced myocardial pro-inflammatory and pro-oxidant cytokines. To detect reactive oxygen species, rats that had undergone 30 min of ischemia were intravenously administered the vehicle or H2-MBs before 40 min of reperfusion. The levels of all of the examined myocardial pro-inflammatory and pro-oxidant cytokines increased after myocardial ischemia-reperfusion, but they decreased by various degrees when the rats received the H2-MBs. (a-c) The levels of pro-inflammatory cytokines, including MPO, TNF-α and IL-1β, were then detected (n = 6). *P < 0.05, **P < 0.01. The data represent the means ± s.d. (d-e) The levels of the pro-oxidant 8-OHdG and of MDA cytokines were detected (n = 6). *P < 28


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0.05, **P < 0.01. The data represent the means ± s.d. (f-i) The levels of radicals (•OH, NO•, O2-•) and H2O2 in the homogenates were detected by measuring their reactivity with HPF, DAF-2, NBT, or CM-H2DCFDA, respectively. The fluorescent intensities were determined using an automatic microplate reader, as described in the methods section (n = 3). *P < 0.05, **P < 0.01. The data represent the means ± s.d.

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Figure 1

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Figure 3

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Figure 5

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Image-Guided Hydrogen Gas Delivery for Protection from Myocardial

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